Binding assays are a frequently used diagnostic tool in the biochemical and medicinal arts. Such assays can provide sensitive probes for determining the presence of small amounts, e.g. nanograms per milliliter, of an entity being assayed.
Binding assays depend upon the interaction or aggregation between two or more entities to form a complex. The presence of the complex is indicated by a label or tag that can be linked to one of the complexed entities or to an exogenously supplied entity that binds to the complex.
One of the most frequently used binding assays is an immunoassay in which one entity is an antigenic protein ligand and the other is a binding reagent such as a receptor comprising an antibody or the idiotype containing portion thereof. The most widely used immunoassay label is a radioisotope [Yalow et al., Nature, 184, 1648-1649 (1959)].
However, radioimmunoassays possess a number of disadvantages. For example, the most commonly used radiolabel, iodine-125, is a gamma-emitting isotope with a relatively short shelf-life that usually requires that the labeled diagnostic be prepared within a few days of its use. Radioisotopes also present health hazards in their preparation and use, and are acoompanied by the production of radioactive wastes that present an ever increasing problem in disposal. Furthermore, radioimmunoassays require the use of expensive and complicated equipment for quantitative measurement of the amount of binding that has occurred.
These defects in the use of radiolabels have prompted many searches for alternative labels. See, for example, the review and references therein by O'Sullivan et al., Annals Clinical Biochem., 16, 221-240 (1979). The most popular of the alternatives to radiolabels thus far utilized are the enzyme labels.
The properties of enzymes allow them to be used in relatively small quantities because their presence is amplified by the generation of multiple quantities of the product of the reaction catalyzed by the enzyme label. This product or indicia, commonly a colored or fluorescent substance, can typically be detected visually, and its amount can be quantified by relatively inexpensive equipment.
Enzymes, however, are difficult to obtain in high purity, are relatively unstable at elevated temperatures, and are also generally costly. In addition, because of their relatively high molecular weight, e.g. about 20,000 to about 100,000 daltons, and concomitant large bulk, only a few enzyme molecules can typically be bonded per molecule of binding reagent such as an antibody without inhibiting the binding ability of the antibody.
Furthermore, to be used as a label in an immunoassay, enzymes must be conjugated or coupled with the antibody. The coupling reactions necessary to perform that conjugation are, however, not clean-cut due to the multiplicity of reactive sites on both the enzyme and the antibody. Such conjugation reactions typically generate large quantities of undesirable, extraneous contaminants that must be removed or otherwise accounted for to produce an enzyme preparation of high activity.
Thus, it would be beneficial, if a label could be found for binding assays generally, and immunoassays in particular, that is inexpensive to produce, utilizes low cost equipment for making quantitative assay determinations, is stable under a wide variety of temperature and solution conditions, can be conjugated to a binding reagent through one or a few sites of reactivity, and also can be used in relatively small quantities because of an amplification of the assay indicia that it produces.